Process for preparing vitamin b{11 -glucoside

ABSTRACT

The present invention relates to a process for preparing vitamin B2-glucoside starting from a oligosaccharide having a glucoside linkage, such as maltose, liquefied starch or sucrose and the like, as the raw material using the microbial cell or the enzyme contained therein (trans glucosidase) of the species of Mucor.

United States Patent Suzuki [451 June 13, 1972 PROCESS FOR PREPARINGVITAMIN B -GLUCOSIDE [72] Inventor: Yukio Suzuki, Kurashiki, Japan [73]Assignee: Hayashibara Company, Okayama-shi,

Okayama, Japan [22] Filed: April 14, 1970 [2]] App]. No.: 28,511

[52] US. Cl. 195/28 R, 195/62 [5 1] Int. Cl. ..Cl2d 5/04 58] Field ofSearch 195/28 [56] References Cited OTHER PUBLICATIONS Whitby,Biochemical Journal, Vol. 50, Pages 433- 438, (1952) PrimaryExaminer-Alvin E. Tanenholtz Attorney--Browdy and Neimark 57 ABSTRACTThe present invention relates to a process for preparing vitamin B-glucoside starting from a oligosaccharide having a glucoside linkage,such as maltose, liquefied starch or sucrose and the like, as the rawmaterial using the microbial cell or the enzyme contained therein (transglucosidase) of the species of Mucor.

6 Claims, No Drawings 1: r ocEss FQR'PREPARTNG VITAMIN B -GLUCOSIDE.

The present invention relates to a process for preparing vitamin B-glucoside starting from oligosaccharide having a glucoside linkage,such as maltose, liquefied starch or sucrose and the like, as therawmaterial using the microbial cell or the enzyme contained therein'(transglucosidase) of a species of Mucor.

Vitamin B was isolated by Kuhn in 1933 as a yellow crystalline pigment.The said vitamin has been regarded as a significant growth factor foranimals, being Synthesized by a biochemical method since then but nowprepared exclusively by a chemical synthesis. j

A tremendous amount of vitamin B has been adopted as a medicine, anedible dye and also utilized as a food additive for domestic animals.However its solubility in water is extremely low, requiring a number ofdevices in order to overcome the difficulty indirect application; Incontrast, vitamin B -glucoside which possesses the same functions asvitamin B is water-soluble and so the industrial preparation thereof hasbeen desired. I i

.The said vitamin B -glucoside was discovered by .L. G. Whitby in 1952as a novel derivative of vitamin B in the course ofstudies on metabolismof thevitamim it has been revealed to play an important role in thematerial metabolic system in the living body (L. G. Whitby,The-Biochemical Journal, Vol.- 50, p.433, 1952). l

Since thereport of L. G. Whitby, the vitamin B -glucosideproducingenzyme (trans-glucosidase, i.e. the glucoside transfer enzyme effectingtransfer of the glucoside group in a diose to vitamin B has beenstudiedby' many investigators and hitherto revealed to be distributedwidely in plant and animal tissues and in microorganisms. However theactivity of the enzyme except that existing in Leuconostoc has beenfound to be rather low.

A process for producing said vitamin B -glucoside utilizing the bacteriawhich refers to Leuconostoc has been proposed (Vitamin, Vol. 23', p. 74,1961;.Iournal of Vitaminology, Vol.

therefrom to obtain the desired product in high yield.

Nevertheless, the separation of the desired product has been with aconversion of 40-95 percent on cultivation with shaking for 2-4 days,and 40-90 percent on cultivation without shaking for 7-10 days.

As mentioned above, the present invention can also be realized by usingthe culture broth filtrate, microbial cells or the enzyme isolatedtherefrom.

The present invention will be further illustrated by the followingexamples. I e

, EXAMPLE 1 To 50 m1 of a culture medium comprising maltose 4 percent,ammonium nitrate 0.1 percent, potassium dihydrogen phosphate 0.1percent, potassium chloride 0.05 percent, calcium carbonate 1 percent(sterilized by heating separately and added during inoculation) wasadded vitamin B, mg, and inoculated with Mucorjavanicus lFO 4569,incubated at C for 2 days with shaking to accumulate 32.8 mg of vitaminB glucoside and minor amount of vitamin B -oligosaccharide. The yield ofthe vitamin B -glucoside versus the used vitamin B was 92.0 percent. Theproduct was adsorbed on Florisil column, eluted with an aqueous pyridinesolution,

precipitated with ethanol after concentration to' give crystals 'ratherdifficult due to formation of p'olysaccharide inevitably as by-products.

ln-consequence of an extensive study on the process for preparingvitamin B -glucoside, the present inventor has been able to achievean'efficient novel process for preparing the said vitamin B -glucosidein high yield based'on the findings that the microorganisms of Mucorspecies effect glucoside transfer from maltose or liquefied starch andthe like to vitamin B during the course of growth. The same glucosicletransfer from maltose or liquefied starch to vitamin 'B, takes placelikewise on using the culture broth filtrate of the Said microorganisms,cell extract or the enzyme therefrom. The activity of said microorganismwere found to be extremely high, and no appreciable amount ofcontaminants such as polysaccharide were accumulated during the process.I

The molds to be utilized in the present invention include the strains ofMucorjavanicus'lFO 4569, 4570, IAM 6101, 6087, Mucor racemosus IAM 6123,IFO 4581, Mucor rowrianus lFO isolated 5773, 1AM 6131, Mucor muceda1505776 and the like, all belong to the genus of Mucor.

' The present invention can be'realized by inoculating one of the abovespecies of microorganisms into a usual culture ture medium as describedin Example 1 to which 15 mg of vitamin B had been added. On-cultivationat 30 C for 3 days with shaking afforded 18.3 mgof vitamin B -glucoside.The yield was 85.0 percent.

' EXAMPLE 3 g T050 ml of the same culture medium as given in Example '1was added 15 mg of vitamin B and subsequently, inoculated with Mucorjavanicus 1E0 4570, cultivated at 30 C for 10 days withoutshaking;.affording 18.5 mg of vitamin B -glucoside. The .yield was 86.5percent. v

. EXAMPLE 4 i Mucor rouxianus IAM 6131 wasused instead .of Mucorracernosus; cultivation of the mold in the same condition ad adoptedinExample 3"for 7 days without shaking afforded 16.0 mg of vitamin B-glucoside. The yield was 75.0 percent.

, EXAMPLE 5 .Mucor javanicus was inoculated into 100 m1 of a culturemedium comprising glucose 3 percent, ammonium nitrate 0.1percent,.sodium nitrate 0.1 percent, potassium dihydrogen phosphate 0.1percent, potassium chloride 0.05 percent and calcium carbonate 1 percentpreviously sterilized by heating separately (to be added during theinoculation), cultivatedat 30 C for 7 days without shaking. Themicrobial cells were collected-and a 25 ml cell suspension was preparedtherefrom with or withoutgrinding, added to a solution containingmaltose '10 g, vitamin B 20 mg, 0.05 M acetate buffer (PH 4.6) 25 ml, asmall amount of toluene and made up to 100 ml with pure water, incubatedat 45 C for 24 hours which resulted in 36.9 mg of vitamin B -glucosideand a Small amount of vitamin B -oligosaccharide.

. v EXAMPLE 6 Mucor javanicus IAM 6101 was inoculated into 100 ml of aculture mediumcomprising powdered-corn starch 2 percent, 7 ammoniumsulfate 0.2 percent, potassium dihydrogen phosphate 0.1 percent,ferroussulfate 0.001 percent, calcium carbonate (sterilized previously andadded to the medium during the inoculation), cultivated at 30 C for 2days with shak-,

ing; the culture broth afterremoval of the microbial cells was used asthe enzyme source. On incubating the reaction mix-.

tained, amounting to a yield of 74.0 percent.

EXAMPLE 7 After addition of 15 mg vitamin B -glucoside to 50 percent Iculture medium with a composition of 3 percentdextrin, 0.1

percentammonium nitrate, 0.1 percent sodiumnitrate, 0.1

. 3 percent potassium phosphate, 0.05' percent potassium chloride, 1percent calcium carbonate (which was separately sterilized by dryheating beforehand, and added at inoculation),-strains of Mucorracemosus 1AM 6123 were inoculated on the medium and subjected toshaking cultivation at 30 C for a period of 3 days to form 7.7 mgvitamin B,- glucoside. Yield was 35.7 percent." i

' EXAMPLE 8 I As a substitute for strains of Mucor racemosus, strains ofMucor rouxianus 1AM 6131 (ATCC mucor ouxic) were used and cultivatedunder the conditions similar to Example 7. Subsequently, 6.5 mg vitaminB -glucoside was formed with a yield of 30.0 percent.

EXAMPLE 9 On a 50 ml culture medium comprising 4 percent potato starch,0.1 percent ammonium nitrate, 0.1 percent sodium nitrate, 0.1 percentpotassium phosphate, 0.05 percent potassium chloride, 0.1 percentpolypeptone, 1 percent calcium carbonate (which was separatelysterilized beforehand, and added at inoculation)'were added strains ofMucor javanicus IFO 4569. After shaking cultivation at 30 C, for aperiod of 4 days, the resultant cells were treated with ultra sonics. Areaction mixture was prepared by diluting the following in pure water tobe a total volume of 100 ml; 5 g soluble starch, 30 mg vitamin'B 33 ml0.05 M acetic acid buffer (PH 4.6), 25 ml ultra sonic treated celldispersion. The reaction mixture was shaken at 37 C for 6 hours. Fromthe reaction mixture 10.7 mg of vitamin Bg-glucoside was obtained. Theyield was 25.0

percent.

EXAMPLE 10,

In replacement of starch, sucrose was used. Cells were collected underthe conditions described in Example 9. Reaction was carried outsimilarly as in Example 9. 12.1 mg vitamin B glucoside was obtained andthe yield was 28.3 percent.

It willbe obvious to those skilled in the-art that various changes maybe made without departing from the scope of the invention and theinvention is not to be considered limited to what is described in thespecification. Y I

What is claimed is: v V v l. A process for preparing vitamin B glucosidewhich comprises treating a solution containing vitamin B, and anoliogosaccharide selected from the group consisting of maltose, sucrose,and liquefied starch with the transglucosidase enzyme isolated from aspecies of Mucar.

2. A process according to claim 1, wherein the said isolated enzyme froma species of Mucor is produced by inoculating and cultivating thespecies of Mucor in culture media containing said oligosaccharide.

3. A process according to claim 1, wherein the said enzyme isolated froma species of Mucor is a fungous enzyme produced by cultivating thespecies of Mucor in usual culture media.

4. A process according to claim 1, wherein the said enzyme isolated froma species of Mucor is the enzyme solution obtained from the culturebroth filtrate produced by cultivating the species of Mucor.

5. A process according to claim 1 wherein the said enzyme isolated froma species of Mucor is the fungus produced by cultivating the species ofMucor and the transfer enzyme separated from the culture broth filtrate.

6. A process according to claim 1, wherein said species of Mucor is oneof the Mucor species of Mucor javanicus (IFO 4569, 4570, 1AM 6101,6087), Mucor racemosus (lFO 4581, 1AM 6l23),-Muc0r rourianus (IFO 5773,1AM 6131), and Mucor mucedo (lFO 5776).

2. A process according to claim 1, wherein the said isolated enzyme froma species of Mucor is produced by inoculating and cultivating thespecies of Mucor in culture media containing said oligosaccharide.
 3. Aprocess according to claim 1, wherein the said enzyme isolated from aspecies of Mucor is a fungous enzyme produced by cultivating the speciesof Mucor in usual culture media.
 4. A process according to claim 1,wherein the said enzyme isolated from a species of Mucor is the enzymesolution obtained from the culture broth filtrate produced bycultivating the species of Mucor.
 5. A process according to claim 1wherein the said enzyme isolated from a species of Mucor is the fungusproduced by cultivating the species of Mucor and the transfer enzymeseparated from the culture broth filtrate.
 6. A process according toclaim 1, wherein said species of Mucor is one of the Mucor species ofMucor javanicus (IFO 4569, 4570, IAM 6101, 6087), Mucor racemosus (IFO4581, IAM 6123), Mucor rourianus (IFO 5773, IAM 6131), and Mucor mucedo(IFO 5776).